Multiple Sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) in which the predominant pathologic findings are demyelination accompanied by disruption of underlying axons (Trapp et al., New Engl. J. Med. 338:278-285, 1998; Prineas, J. W., xe2x80x9cPathology of Multiple Sclerosisxe2x80x9d in: Cook SD, ed. Handbook of Multiple Sclerosis. New York: Marcel Dekker, Inc, 1990:187-215). The disease affects young adults who usually present with a relapsing, remitting pattern of neurologic involvement and progress to a chronic phase with increasing difficulty in ambulation and coordination. The etiology of MS is not known, but there is considerable indirect evidence that argues for the role of an infectious agent(s) in the pathogenesis of the disease. Epidemiological studies strongly suggest that a CNS infection in early childhood is a key factor in the development of MS (Kurtzke, Clin. Microbiol. Rev. 6:382-427, 1993). Viral infections have long been thought to play a possible role in the pathogenesis of MS because viruses are known to cause demyelinating disease in experimental animals, often present clinically with relapsing, remitting symptoms, and can cause disease with long periods of latency (Cook and Dowling, Neurology 30:80-91, 1980; Johnson, R. T., Viral infections of the Nervous System. New York: Raven Press, 1982). Studies to date, however, have failed to identify any virus as playing a major role in MS, although activated human herpes virus 6 (HHV-6) has been identified recently in brains of MS patients (Sanders et al., J. Neurovirol. 2:249-258, 1996; Challoner et al., Proc. Natl. Acad. Sci. USA 92:7440-7444, 1995; Merelli, J. Neurol. 244:450-454, 1997). Although an immune response to this virus is seen during acute exacerbations, the role of HHV-6 infection in MS remains unclear (Soldan et al., Nature Med. 3:1394-1397, 1997).
Current opinion thus favors MS to be an autoimmune disease directed against self neural antigens (Martin et al., Annu. Rev. Immunol. 10:153-169, 1992). To reconcile the role of environment in the pathogenesis of MS as well as the absence of an identifiable infectious pathogen, it is believed that infectious agents may act to trigger an autoimmune process. Such an autoimmune response may result from structural similarities between an infectious agent and neural antigens (antigenic mimicry) or from an expansion of self autoreactive T cell clones in response to bacterial or viral superantigens (Brocke et al., Nature 65:642-646, 1993; Jahnke et al., Science 229:282-284, 1985; Marrack and Kappler, Science 248:325-329, 1998; Oldstone, J. Autoimmun. 2(S):187-194, 1989). Evidence that MS is a disease mediated by T cells that recognize neural antigens has been hard to justify, since measures directed at either eliminating or reducing helper T cell function have not changed the natural history of MS (Sriram and Rodriguez, Neurology 48:469-473, 1997). Improved methods of diagnosing MS would facilitate identification of treatable pathogens and expedite commencement of treatment.
Over the last few years, therapy with xcex2-IFN has emerged as a means of reducing the morbidity of MS. Both xcex2-IFNs (xcex2-1a and xcex2-1b) reduce the number of clinical relapses and slow the progression of the disease. In addition, magnetic resonance imaging (MRI) studies demonstrate a decrease in the number of new inflammatory cerebral lesions in patients receiving xcex2-IFN. Although xcex2-IFN was introduced as a therapeutic agent for MS based on its anti-viral properties, the reasons for the therapeutic benefit of xcex2-IFN for MS remain unclear. Thus far, no viral agent has been consistently found to be associated with MS.
In a first aspect, the present invention features a method of diagnosing or monitoring multiple sclerosis in an individual, including assaying a test sample from the individual for the presence of Chlamydia, wherein the presence of Chlamydia in the sample indicates the presence of multiple sclerosis.
In preferred embodiments, the Chlamydia is selected from the group consisting of Chlamydia pneumoniae, Chlamydia pecorum, Chlamydia psittacci, and Chlamydia trachomatis, and the test sample is selected from the group consisting of blood, serum, peripheral blood mononuclear cells, cerebrospinal fluid, urine, nasal secretion, and saliva.
In one embodiment, the test sample is assayed for the presence of Chiamydia by contacting cultured chlamydia-free indicator cells (e.g., HL cells, H292 cells, HeLa cells, or Hep-2 cells) with the test sample; and then detecting the presence of Chlamydia in the cultured indicator cells. The presence of Chlamydia in the cultured indicator cells is indicative of the presence of Chlamydia in the test sample.
The presence of Chlamydia in the cultured indicator cells can be detected by detecting an antibody to Chlamydia (e.g, an antibody to a Chlamydia elementary body antigen), a Chlamydia gene, or a Chlamydia protein in the test sample. The presence of the antibody, gene, or protein is indicative of the presence of Chlamydia in the test sample. In one embodiment, the test sample is incubated under disulfide reducing conditions (e.g., incubating a disulfide reducing agent such as 2,3-dimercaptosuccinic acid, penicillamine, xcex2-lactams, dithiotreitol, mercaptoethylamine, or N-acetylcysteine) prior to detecting the presence of Chlamydia.
In another aspect, the invention features a method of isolating elementary bodies from a receptacle containing elementary bodies. The method includes treating the receptable with trypsin/EDTA to release elementary bodies adhered to the receptacle; and then concentrating the elementary bodies by centrifugation or filtration.
In still another aspect, the invention features a method of releasing DNA from elementary bodies, the method including incubating the elementary bodies under disulfide reducing conditions and digesting the elementary bodies with a protease.
In yet another aspect, the invention features a method of treating an individual diagnosed to have multiple sclerosis, including administering to the individual an effective amount of at least one anti-chlamydial agent. In one embodiment, the individual is administered the anti-chlamydial agent until the individual tests negative for elementary body phase Chlamydia, replicating phase Chlamydia, and cryptic phase Chlamydia. In another aspect, the individual is administered the anti-chlamydial agent for at least 45 days. The adminstration can be continued for longer periods, and it may be preferable to continue the treatment for at least 90 days, at least 180 days, or even for one year or more.
Preferable anti-chlamydial agents include rifamycins, azalides, macrolides, ketolides, streptogramins, ampicillin, amoxicillin, nitroimidazoles, nitrofurans, quilolones, fluoroquinolones, sulfonamides, isonicotinic congeners, and tetracyclines.
In one embodiment, the individual is also administered an effective amount of an agent that increases inducible nitric oxide synthase (iNOS) activity, such as a type-1 interferon (e.g., xcex1-interferon or xcex2-interferon), a synthetic type-1 interferon analog, or a hybrid type-1 interferon. Preferably, the type-1 interferon analog or hybrid binds to the same receptor as a naturally-occurring type-1 interferon. In another embodiment, the individual is administered at least two anti-chlamydial agents.
In yet another aspect, the invention features a method of treating an individual diagnosed to have multiple sclerosis, including administering to the individual (i) a rifamycin; and (ii) a compound selected from the group consisting of azalides, macrolides, ketolides, and streptogramins. In addition, the individual can optionally be administered ampicillin, amoxicillin, probenecid, a nitroimidazole, a nitrofuran, or any combination thereof.
In another aspect, the invention features a method of treating an individual diagnosed to have multiple sclerosis, including administering to the individual one of the following combinations: a rifamycin, ampicillin or amoxicillin, and probenecid; a quinolone or a fluoroquinolone and a rifamycin; a rifamycin, a sulfonamide, and an isonitotinic congener; or a rifamycin and a tetracycline. The individual can also be administered an effective amount of a compound that increases iNOS activity (e.g., xcex2-interferon).
The administration is preferably continued until the individual tests negative for elementary body phase Chlamydia, replicating phase Chlamydia, and cryptic phase Chlamydia, or for at least 45 days.
In still another aspect, the invention features a pharmaceutical composition that includes one of the following combinations: a rifamycin, ampicillin or amoxicillin, and probenecid; a quinolone or a fluoroquinolone and a rifamycin; a rifamycin, a sulfonamide, and an isonitotinic congener; or a rifamycin and a tetracycline. The composition can optionally include a compound that increases iNOS activity (e.g., xcex2-interferon).
In yet another aspect, the invention features a kit that includes an anti-chlamydial agent and a compound that increases iNOS activity. In a preferred embodiment, the compound that increases iNOS activity is a type-1 interferon (e.g., xcex2-interferon), a synthetic type-1 interferon analog, or a hybrid type-1 interferon, wherein the type-1 interferon analog or hybrid binds to the same receptor as a naturally-occurring type-1 interferon. In another preferred embodiment, the anti-chlamydial agent is selected from the group consisting of rifamycins, azalides, macrolides, ketolides, streptogramins, ampicillin, amoxicillin, nitroimidazoles, quilolones, fluoroquinolones, sulfonamides, isonicotinic congeners, and tetracyclines.
In still another aspect, the invention features a method for determining whether a candidate compound is a potential drug for the treatment of a disease caused or exacerbated by chlamydial infection, the method including the steps of: (a) infecting a non-human animal (e.g., a non-human mammal) with Chiamydia; (b) administering a candidate compound to the animal; and (c) assaying for the presence of a chlamydial infection in a test sample from the mammal. A decrease in the level of infection, relative to the level of infection of a control animal infected with chlamydia but not administered a candidate compound, identifies the candidate compound as a potential drug for the treatment of disease caused or exacerbated by a chlamydial infection. Preferably, the animal is a non-human mammal and brain of the mammal is infected with Chlamydia.
In preferred embodiments, the Chlamydia is selected from the group consisting of Chlamydia pneumoniae, Chlamydia pecorum, Chlamydia psittacci, and Chlamydia trachomatis, and the test sample is selected from the group consisting of blood, serum, cerebrospinal fluid, urine, nasal secretion, and saliva. In another preferred embodiment, the disease is multiple sclerosis. The animal can be, for example, a mouse, rat, rabbit, or amoeba.
In one embodiment, the test sample is assayed for the presence of Chlamydia by contacting cultured chlamydia-free indicator cells (e.g., HL cells, H292 cells, HeLa cells, or Hep-2 cells) with the test sample; and then detecting the presence of Chlamydia in the cultured indicator cells. The presence of Chlamydia in the cultured indicator cells is indicative of the presence of Chlamydia in the test sample.
The presence of Chlamydia in the cultured indicator cells can also be detected by detecting an antibody to Chlamydia (e.g, an antibody to a Chlamydia elementary body antigen), a Chlamydia gene, or a Chlamydia protein in the test sample. The presence of the antibody, gene, or protein is indicative of the presence of Chlamydia in the test sample. In one embodiment, the test sample is incubated under disulfide reducing conditions (e.g., incubating a disulfide reducing agent such as 2,3-dimercaptosuccinic acid, penicillamine, xcex2-lactams, dithiotreitol, mercaptoethylamine, or N-acetylcysteine) prior to detecting the presence of Chlamydia.
In a related aspect, the invention features a second method for determining whether a candidate compound is a potential drug for the treatment of multiple sclerosis. This method includes the steps of: (a) infecting the brain of a non-human mammal (e.g., a rat, mouse, or rabbit) with Chlamydia; (b) administering a candidate compound to the mammal; and (c) assaying for the loss of white matter in the brain of the mammal, wherein a decrease in the loss of white matter, relative to the loss of white matter in a control mammal infected with chlamydia but not administered any candidate compound, identifies the candidate compound as a potential drug for the treatment of multiple sclerosis.
By xe2x80x9cChlamydiaxe2x80x9d or xe2x80x9cchlamydial cellxe2x80x9d is meant any organism of the order Chlamydiales. Examples include, but are not limited to, C. psittacci, C. trachomatis, C. pecorum, C. abortus, C. caviae, C. felis, C. suis, C. muridarum, WSU-86-1044, Parachlamydia acanthamoebae, and Simkania negevensis. By xe2x80x9cchlamydial infectionxe2x80x9d is meant an infection of a cell by a chlamydial cell.
By xe2x80x9cindicator cellxe2x80x9d is meant a cell capable of being infected by a Chlamydia cell. Preferred indicator cells include HL cells, H292 cells, HeLa cells, and Hep-2 cells, which have been shown to be free of chlamydial infection.
By xe2x80x9clong-term therapyxe2x80x9d is meant the treatment of a disease (e.g., MS) for at least 45 days, more preferably for at least 60 days or even 90 days, and most preferably for at least 120 days, 180 days, or for a year or more. The long-term therapy can be continued for a given length, or can be stopped when a patient tests negative for elementary body phase Chlamydia, replicating phase Chlamydia, and cryptic phase Chlamydia (e.g., by PCR of a disulfide reducing agent-treated sample from the patient).
It may be desirable to change one or all of the drugs in the middle of the long-term therapy. Changes in drug combinations may be for many reasons, such as to reduce side effects or cost to the patient, or in response to a change in the patient""s condition or degree of infection. Moreover, while it is preferable that the therapy is continuous, it is understood that interruption for as much as two weeks or even a month may be desirable or necessary. For example, an individual may take drug combination A for 30 days, stop therapy for two weeks, and then resume therapy (switching to drug combination B) for an additional 30 days. Interrupted therapy and therapy in which one or more drugs are added or removed are each considered to be long-term therapy if the number of days of therapy (i.e., excluding the days in which no drugs for the treatment of MS were administered) is at least 45.
By xe2x80x9canti-chlamydial agentxe2x80x9d is meant an agent that results in a decrease in the viability or replication of chlamydial cells at a concentration that would not be substantially detrimental to the cells in which the chlamydial cells were contained. Preferably, the anti-chlamydial agent decreases the viability or replication of chlamydial cells by at least 50%, more preferably by at least 75% and most preferably by at least 90% or even 95%. Preferred anti-chlamydial agents include, without limitation, rifamycins, azalides, macrolides, ketolides, streptogramins, ampicillin, amoxicillin, nitroimidazoles, quilolones, fluoroquinolones, sulfonamides, isonicotinic congeners, and tetracyclines.
The present invention provides methods for the diagnosis of MS with a significant reduction in cost. In addition, these diagnostic assays provide objective data concerning the course of the disease and, thus, the ability to monitor disease progress and the effectiveness of therapy. The invention also provides methods and reagents for the treatment of a patient diagnosed with MS, as well as methods for identifying new drugs for the such treatment.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.